Gene expression phenotypes were evaluated for intramuscular fat (IMF) in bovine skeletal muscle as an alternative to traditional estimates of IMF%. Gene expression data from a time course of LM development in high- and low-marbling Bos taurus cattle crosses were compared to identify genes involved in intramuscular adipocyte lipid metabolism with developmentally similar gene expression profiles. Three sets of genes were identified: triacylglyceride (TAG) synthesis and storage, fatty acid (FA) synthesis, and PPARγ-related genes. In an independent analysis in the LM of 48 Bos indicus cattle, TAG and FA gene sets were enriched in the top 100 genes of which expression was most correlated with IMF% (P = 1.2 × 10(-24) and 3.5 × 10(-9), respectively). In general, genes encoding enzymes involved in the synthesis of FA and TAG in the intramuscular adipocytes were present in the top 100 genes. In B. indicus, effects of a steroid hormone growth promotant (HGP), 2 experimental sites [New South Wales (NSW) and Western Australia (WA)], and 3 tenderness genotypes on the expression levels of genes in the TAG gene set and the correlation of gene expression with IMF% were investigated. Although correlation between expression of 12 individual TAG genes and IMF% was observed in HGP-treated animals in both experimental sites (mean r = 0.43), correlation was not observed for untreated animals at the NSW site (mean r = -0.07, P < 3 × 10(-6)). However, TAG genes showed an average 1.6-fold (P < 0.0004) reduction in expression in the LM of HGP-treated cattle relative to untreated cattle, an effect consistent across both experimental sites. Cattle possessing the favored tenderness calpain 1 and 3 and calpastatin alleles exhibited a greater (P = 0.008) reduction in expression in NSW (1.8-fold reduction, P = 0.0002) compared with WA (1.2-fold reduction, P = 0.03). Tenderness genotype had no impact (P > 0.05) on the correlation of TAG genes with IMF%. In general, the interactions among genotype, treatment and location, and TAG gene set gene expression were consistent with the interactions among the same factors and IMF% detected using 255 animals, of which the 48 in this study were a subset. Thus, the TAG gene set constitutes a gene expression phenotype able to predict effects of different genotypes and treatments on IMF% using much smaller groups than current approaches, even in animals with very low IMF%.